Novel EM Guided Endovascular Instrumentation for In Situ Endograft Fenestration.

Objective: This work aims at providing novel endovascular instrumentation to overcome current technical limitations of in situ endograft fenestration including challenges in targeting the fenestration site under fluoroscopic control and supplying mechanical support during endograft perforation. Technology: Novel electromagnetically trackable instruments were developed to facilitate the navigation of the fenestration device and its stabilization at the target site. In vitro trials were performed to preliminary evaluate the proposed instrumentation for the antegrade in situ fenestration of an aortic endograft, using a laser guidewire designed ad hoc and the sharp end of a commercial endovascular guidewire. Results: In situ fenestration was successfully performed in 22 trials. A total of two laser tools were employed since an over bending of laser guidewire tip, due to its manufacturing, caused the damage of the sensor in the first device used. Conclusions: Preliminary in vitro trials demonstrate the feasibility of the proposed instrumentation which could widespread the procedure for in situ fenestration. The results obtained should be validated performing animal studies. Clinical Impact: The proposed instrumentation has the potential to expand indications for standard endovascular aneurysm repair to cases of acute syndromes.

Vaccine allergy evaluation and management at the specialized Green Channel Consultation Clinic.

BACKGROUND: Suspected vaccine allergy may be a cause of incomplete or delayed vaccination. Patients at risk of adverse reactions or suspected contraindications need specialized consultation about subsequent vaccinations. OBJECTIVE: To analyse consultancy results for patients at risk of allergic reactions to vaccines as evaluated by the Green Channel University Hospital Immunization Consultancy Clinic. METHODS: A review of cases of allergic reactions to vaccines or contraindications due to underlying diseases or sensitization to vaccine components submitted to the Green Channel was carried out. Analysed data included detailed clinical reaction history, skin and in vitro allergy testing with vaccine components, recommendations for vaccination and outcome of subsequent vaccine administrations. RESULTS: A total of 519 cases, 370 referred for previous local or systemic reactions to vaccines, mostly cutaneous, and 149 sent for suspected contraindications were evaluated. Skin testing was performed on 152 patients, specific IgE determination in 37 subjects and patch testing in 173 cases. After consultation, 442 (85%) subjects were advised to continue vaccination, with personalized precautions (premedication, or alternative brand, or administration in graded doses) for 200 of them. Among the 352 (80%) patients vaccinated as per Green Channel instructions, 33 subjects (9.3%) reported mild allergic or non-specific symptoms and one (0.3%) urticaria with bronchospasm. CONCLUSION AND CLINICAL RELEVANCE: Even though vaccine allergy occurs very rarely, a safe procedure for immunization can be applied, through specialized allergy consultancy, for most subjects with suspected allergy to vaccines, and who could be potentially excluded from vaccination for risk of adverse reactions.

Peptide analogues of a subdominant epitope expressed in ebv-associated tumors: synthesis and immunological activity.

H-Cys-Leu-Gly-Gly-Leu-Leu-Thr-Met-Val-OH (CLG) peptide is an EBV subdominant epitope that represents the target of HLA-A2 restricted CTL responses. The CLG peptide has low affinity for HLA-A2 and does not produce stable complexes, both factors that determine weak CTL responses. In contrast, the [Tyr(1), Ala(3)]CLG (YLA) analogue showed high affinity for HLA-A2 molecules and efficiently stimulated CLG-specific CTL precursors. Nevertheless, this modified epitope showed low enzymatic stability. To further improve the immunotherapeutical potential of this "improved epitope", we have synthesized and tested YLA analogues containing different modifications next to the scissile peptide bond. Among the analogues we found three peptides, with higher enzymatic resistance, that efficiently stimulate CTL responses. These peptides may be used for EBV-specific immunotherapies.

Supra-agonist peptides enhance the reactivation of memory CTL responses.

Single amino acid substitutions at TCR contacts may transform a natural peptide Ag in CTL ligands with partial agonist, antagonist, or null activity. We obtained peptide variants by changing nonanchor amino acid residues involved in MHC class I binding. These peptides were derived from a subdominant HLA-A2-presented, latent membrane protein 2-derived epitope expressed in EBV-infected cells and in EBV-associated tumors. We found that small structural changes produced ligands with vastly different activities. In particular, the variants that associated more stably to HLA-A2/molecules did not activate any CTL function, behaving as null ligands. Interestingly, T cell stimulations performed with the combination of null ligands and the natural epitope produced significantly higher specific CTL reactivation than reactivation of CTLs induced by the wild-type epitope alone. In addition, these particular variants activated memory CTL responses in the presence of concentrations of natural epitope that per se did not induce T cell responses. We show here that null ligands increased ZAP-70 tyrosine kinase activation induced by the natural epitope. Our results demonstrate for the first time that particular peptide variants, apparently behaving as null ligands, interact with the TCR, showing a supra-agonist activity. These variant peptides did not affect the effector T cell functions activated by the natural epitope. Supra-agonist peptides represent the counterpart of antagonists and may have important applications in the development of therapeutic peptides.

Design of dimeric peptides obtained from a subdominant Epstein-Barr virus LMP2-derived epitope.

The latent membrane protein 2 (LMP2) is expressed in EBV-associated tumours. LMP2 is a target of HLA-A2 restricted EBV-specific CTL responses and consequently it may represent a good target for specific CTL-based immunotherapies. However, the efficacy of such therapy is limited by the poor immunogenicity of the protein that induces weak cytotoxic T lymphocyte (CTL) responses directed against the CLGGLLTMV (CLG) epitope. Indeed, the CLG peptide presents low affinity for HLA-A2 and does not produce stable complexes. Therefore we synthesized and tested CLG-dimeric analogues with the purpose of characterizing new compounds with the capacity to bind HLA-A2 molecules. By these studies we have identified a few peptides which, compared to the natural epitope, showed higher affinity for HLA-A2 molecules and superior capacity to form a complex. These dimeric peptides may have the potential to induce efficient CTL responses directed to the natural epitope.

Selective amino acid substitutions of a subdominant Epstein-Barr virus LMP2-derived epitope increase HLA/peptide complex stability and immunogenicity: implications for immunotherapy of Epstein-Barr virus-associated malignancies.

The latent membrane protein 2 is an immunogenic antigen expressed in Epstein-Barr virus (EBV)-associated tumors and consequently it may represent a target for specific cytotoxic T lymphocyte (CTL)-based immunotherapies. However, the efficacy of such a therapy is limited by the poor immunogenicity of the protein that induces weak CTL responses directed to the CLGGLLTMV (CLG) epitope only in the minority of EBV-seropositive donors. We have now demonstrated that selective peptide stimulation of peripheral blood lymphocytes induced CLG-specific CTL in all donors, suggesting that this epitope can be a suitable target for specific immunotherapies. We found that the CLG peptide has a low affinity for HLA-A*0201 and does not produce stable complexes, both factors that are likely to determine the strength of CTL responses to this epitope. Therefore, we synthesized and tested CLG analogues carrying single or combined amino acid substitutions to increase HLA/peptide stability. Among the analogues tested we identified two peptides which, compared to the natural epitope, showed higher affinity for HLA-A*0201 molecules, and produced stable complexes. These peptides demonstrated a potent, specific stimulatory capacity and could be used for selective CTL-based therapies.

The lifespan of major histocompatibility complex class I/peptide complexes determines the efficiency of cytotoxic T-lymphocyte responses.

Major histocompatibility complex (MHC)/peptide association and stability are determined by specific amino acid interactions between peptide antigens and the MHC groove, and are regarded as a critical feature in ensuring efficient monitoring by T cells. In this investigation we examined the relationship between MHC/peptide stability and the immunostimulatory capacity of MHC/peptide complexes. For this purpose we compared synthetic peptide analogues derived from the immunodominant HLA-A11-presented IVTDFSVIK (IVT) epitope, for their capacity to reactivate IVT-specific memory cytotoxic T-lymphocyte (CTL) responses. The analogues differentiated from the wild-type epitope by single amino acid substitution at position 2. All peptides showed similar affinity for HLA-A11 molecules and were recognized by IVT-specific CTL clones, but induced HLA-A11 complexes at the cell surface with different lifespan. This model offered the possibility of comparing the capacity of an immunogenic epitope to stimulate a unique population of T-cell precursors depending on the lifespan of its presentation at the cell surface. We demonstrated that stable HLA-A11/peptide complexes efficiently stimulate IVT-specific CTL responses, while HLA-A11/peptide complexes with short lifespan do not. The precise identification of the role of amino acid residues in the formation of stable MHC/peptide complexes may be relevant for the design of wild-type-derived epitopes with high immunogenicity. These analogues may have important applications in the immunotherapy of infectious diseases and immunogenic tumours.

Mutations in autosomal dominant polycystic kidney disease 2 gene: Reduced expression of PKD2 protein in lymphoblastoid cells.

The polycystic kidney disease 2 (PKD2) gene, encoding a 968-amino acid integral membrane protein with six predicted membrane-spanning domains and intracellular NH2 and COOH termini, is mutated in approximately 15% of the cases of autosomal dominant polycystic kidney disease (ADPKD), a common genetic disease frequently resulting in renal failure. For a better understanding of the cause of this disorder, we searched for mutations in the PKD2 gene in two PKD2-linked families characterized by different clinical phenotypes. A common polymorphism, a nonsense mutation, and a frameshift mutation were found. Both mutations are predicted to produce truncated proteins of 314 and 386 amino acids, arrested at the first extracellular loop of the protein. Restriction enzyme analysis of polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR products, respectively, showed that mutations cosegregated with the disease and mutated alleles were expressed at the messenger RNA level in lymphoblastoid cell lines. However, in these cells, Western blot analysis showed only PKD2 normal protein, and it was expressed at a lower level than that found in cells without the PKD2 mutation. These findings suggest that in lymphoblastoid cells, the truncated protein product of the mutant allele may not be stable.

A heparin cofactor II mutation (HCII Rimini) combined with factor V Leiden or type I protein C deficiency in two unrelated thrombophilic subjects.

305 patients with juvenile thromboembolic episodes were screened for the presence of heparin cofactor II deficiency. The heterozygous deletion of two bases was found in the exon 5 of the heparin cofactor II gene in two unrelated patients, very likely due to a founder effect. This molecular lesion, causing a frameshift and elongated translation, affects the core of the molecule and should cause the complete unfolding of the protein, which is in accordance with the observed type I deficiency. The corresponding region of antithrombin III gene is affected by a cluster of frameshift mutations suggesting that heparin cofactor II and antithrombin III could share similar mutational patterns. The heparin cofactor II gene alteration was associated with, in one patient, the factor V Leiden mutation and, in the other, type I protein C deficiency. The tracing of the single defects in several family members indicated that the mutations became clinically manifest only when present in the doubly heterozygous condition. This study provides two examples, based on molecular findings, of the interplay of risk factors which is potentially useful to define a role for heparin cofactor II deficiency in inherited thrombophilia.